Related Articles

Type I interferons are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Type I interferon-inducible mRNAs are widely and concordantly overexpressed in the periphery and involved tissues of a subset of SLE patients, and provide utility as pharmacodynamic biomarkers to aid dose selection, as well as potential indicators of patients who might respond favorably to anti-IFNα therapy in SLE. We implemented a three-tiered approach to identify a panel of type I interferon-inducible mRNAs to be used as potential pharmacodynamic biomarkers to aid dose selection in clinical trials of sifalimumab, an anti-IFNα monoclonal antibody under development for the treatment of SLE. In a single-dose escalation phase 1 trial, we observed a sifalimumab-specific and dose-dependent inhibition of the overexpression of type I interferon-inducible mRNAs in the blood of treated subjects. Inhibition of expression of type I interferon-inducible mRNAs and proteins was also observed in skin lesions of SLE subjects from the same trial. Inhibiting IFNα resulted in a profound downstream effect in these SLE subjects that included suppression of mRNAs of B-cell activating factor belonging to the TNF family and the signaling pathways of TNFα, IL-10, IL-1β, and granulocyte-macrophage colony-stimulating factor in both the periphery and skin lesions. A scoring method based on the expression of type I interferon-inducible mRNAs partitioned SLE patients into two distinct subpopulations, which suggests the possibility of using these type I interferon-inducible genes as predictive biomarkers to identify SLE patients who might respond more favorably to anti-type I interferon therapy.

Introduction

Glucocorticoid (GC) therapy is the main treatment for systemic lupus erythematosus (SLE). However, some patients are resistant to these agents. Abnormalities of glucocorticoid receptor (GR) seem to be related to steroid resistance. This study evaluated GRs in T lymphocytes and monocytes of SLE patients by flow cytometry (FCM) using a monoclonal antibody (mAb) and FITC-Dex probes.

Methods

Thirty-five patients with SLE before treatment and 27 age- and sex-matched normal controls were studied. Disease activity scores were determined before and after treatment and used to divide the patients into steroid-resistant (SR) and steroid-sensitive (SS) groups. GRs in T lymphocytes (CD3+) and monocytes (CD14+) were examined by FCM with GR-mAb and FITC-Dex probes before treatment. Peripheral blood mononuclear cells (PBMCs) were isolated for in vitro GCs sensitivity assays. The validity of FCM analysis of intracellular staining for GR with GR-mAb and FITC-Dex probes was evaluated through comparison with western blot and radioligand binding assay (RLBA) in U937 and K562 cells in vitro. One-way ANOVA, student's t test, linear regression and spearman correlation were performed.

Results

A significant decrease in GR binding and the expression in K562 and U937 cells with 10-6 M dexamethasone (Dex) was found compared with those without Dex. In addition, a positive correlation was found between FCM and RLBA as well as FCM and Western blot. The expression and binding of both CD3/GR and CD14/GR in SR patients with SLE, detected by FCM, were all lower than those in SS patients with SLE, whereas there was no significant difference in SS patients and controls. In vitro corticosteroid sensitivity assay indicated that PHA-stimulated tumour necrosis factor-α (TNF-α), IL-12 and interferon-γ (IFN-γ) secretion was significantly inhibited by 10-6 M Dexamethasone in all controls and SS patients, compared with that in SR group, which confirms patient classification as SR and SS by disease activity index (SLEDAI) score.

Conclusions

Abnormalities of expression and binding of the GR may be involved in tissue resistance to steroids in SLE patients. Determination of GR expression and binding by FCM may be useful in predicting the response to steroid treatment of SLE patients.

Trial registration

Clinical trial registration number NCT00600652.

Background

Overexpression of type I interferon (IFN-I)-induced genes is a common feature of systemic lupus erythematosus (SLE) and its experimental models, but the participation of endogenous overproduction of IFN-I on it is not clear. To explore the possibility that abnormally increased IFN-I receptor (IFNAR) signaling could participate in IFN-I-induced gene overexpression of SLE, we examined the phosphorylation status of the IFNAR-associated signaling partners Jak1 and STAT2, and its relation with expression of its physiologic inhibitor SOCS1 and with plasma levels of IFNα and IFN-like activity.

Methodology/Principal Findings

Peripheral blood mononuclear cells (PBMC) from SLE patients with or without disease activity and healthy controls cultured in the presence or in the absence of IFNβ were examined by immunoprecipitation and/or western blotting for expression of the two IFNAR chains, Jak1, Tyk2, and STAT2 and their phosphorylated forms. In SLE but not in healthy control PBMC, Jak1 and STAT2 were constitutively phosphorylated, even in the absence of disease activity (basal pJak1: controls vs. active SLE p<0.0001 and controls vs. inactive SLE p = 0.0006; basal pSTAT2: controls vs. active and inactive SLE p<0.0001). Although SOCS1 protein was slightly but significantly decreased in SLE in the absence or in the presence of IFNβ (p = 0.0096 to p<0.0001), in SOCS1 mRNA levels were markedly decreased (p = 0.036 to p<0.0001). IFNβ induced higher levels of the IFN-I-dependent MxA protein mRNA in SLE than in healthy controls, whereas the opposite was observed for SOCS1. Although there was no relation to increased serum IFNα, active SLE plasma could induce expression of IFN-dependent genes by normal PBMC.

Conclusions/Significance

These findings suggest that in some SLE patients IFN-I dependent gene expression could be the result of a low IFNAR signaling threshold.

Objective

To describe a new systemic lupus erythematosus (SLE) Responder Index (SRI) based on the belimumab phase II SLE trial and demonstrate its potential utility in SLE clinical trials.

Methods

Data from a 449-patient randomized, double-blind, placebo-controlled study of 3 doses of belimumab (1, 4, 10 mg/kg) or placebo plus standard of care therapy (SOC) over a 56-week period were analyzed. SELENA-SLEDAI and BILAG SLE disease activity instruments, SF-36 Health Survey, and biomarker analyses were used to create a novel SRI. Response to treatment in a subset of SLE patients (n=321) who were serologically active (ANA ≥1:80 and/or anti-dsDNA antibody ≥30 IU) at baseline was retrospectively evaluated using the SRI.

Results

SRI response is defined as: 1) ≥4-point reduction in SELENA-SLEDAI score; 2) no new BILAG A or no more than 1 new BILAG B domain score; and 3) no deterioration from baseline in the Physician’s Global Assessment (PGA) by ≥0.3 points. In serologically active patients, addition of belimumab to SOC resulted in a response in 46% of patients at week 52 compared with 29% for the placebo patients (P=0.006). SRI responses were independent of baseline autoantibody subtype.

Conclusion

Evidence-based evaluation of a large randomized, placebo-controlled trial in SLE resulted in the ability to define a robust responder index based on improvement in disease activity without worsening of the overall condition or the development of significant disease activity in new organ systems.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a breakdown of tolerance to nuclear antigens and the development of immune complexes. Genomic approaches have shown that human SLE leukocytes homogeneously express type I interferon (IFN)–induced and neutrophil-related transcripts. Increased production and/or bioavailability of IFN-α and associated alterations in dendritic cell (DC) homeostasis have been linked to lupus pathogenesis. Although neutrophils have long been shown to be associated with lupus, their potential role in disease pathogenesis remains elusive. Here, we show that mature SLE neutrophils are primed in vivo by type I IFN and die upon exposure to SLE-derived anti-ribonucleoprotein antibodies, releasing neutrophil extracellular traps (NETs). SLE NETs contain DNA as well as large amounts of LL37 and HMGB1, neutrophil proteins that facilitate the uptake and recognition of mammalian DNA by plasmacytoid DCs (pDCs). Indeed, SLE NETs activate pDCs to produce high levels of IFN-α in a DNA- and TLR9 (Toll-like receptor 9)–dependent manner. Our results reveal an unsuspected role for neutrophils in SLE pathogenesis and identify a novel link between nucleic acid–recognizing antibodies and type I IFN production in this disease.

Objective

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by unpredictable flares of disease activity and irreversible damage to multiple organ systems. An earlier study showed that SLE patients carrying an interferon gene expression signature in blood have elevated serum levels of interferon (IFN)-regulated chemokines. These chemokines were associated with more severe and active disease and showed promise as SLE disease activity biomarkers. This study was designed to validate IFN regulated chemokines as biomarkers of SLE disease activity in 267 longitudinally-followed SLE patients.

Methods

To validate the potential utility of serum chemokine levels as biomarkers for disease activity, we measured serum chemokine levels – CXCL10 (IP-10), CCL2 (MCP-1), and CCL19 (MIP-3B) – in an independent cohort of 267 SLE patients followed longitudinally over one year (1166 total visits).

Results

Serum chemokine levels correlated with current visit lupus activity (p=2×10−10), rising at flare (p=1×10−3) and decreasing as disease remitted (p=1×10−3), and performed better than currently available laboratory tests. Chemokine levels measured at a single baseline visit in patients with SLEDAI ≤4 were predictive of lupus flare over the ensuing year (p=6×10−4).

Conclusion

Monitoring serum chemokine levels in SLE may improve assessment of current disease activity, the prediction of future flare, and overall clinical decision-making.

Systemic lupus erythematosus (SLE) is a chronic inflammatory disorder that is driven by autoantibodies that target multiple organ systems. B-lymphocyte stimulator (BLyS) and its receptors on B-cell subsets play an important role in autoimmune B-cell development and SLE pathogenesis. Targeted therapy with belimumab, the monoclonal antibody against BLyS, has shown clinical benefit in two large-scale, multicenter phase III trials leading to US Food and Drug Administration approval for patients with serologically positive SLE who have active disease despite standard therapy. This review will discuss the challenges in lupus drug development and clinical trials, the basics of B-cell pathogenesis in SLE, the recent lupus clinical trials of B-cell targeted treatments, and other potential targeted therapies under investigation for patients with lupus.

The IFN-I pathway is activated in systemic lupus erythematosus (SLE) and appears to be important in the pathogenesis of the disease. As a result, several clinical trials of anti-IFN monoclonal antibodies, which hold promise to control the disease, have been launched. Additionally, activation of IFN-I might be important in the prognosis and activity assessment of the disease. Therefore, new biomarkers that reflect activity of the IFN-I pathway and are simple to measure, such as the monocyte CD64 receptor, are expected to have a great impact on the management of SLE, if properly validated.

It is shown in this study that the heparan sulfate proteoglycan agrin is overexpressed in T cells isolated from patients with the autoimmune disease systemic lupus erythematosus (SLE). Freshly isolated CD4+ and CD8+ subpopulations both exhibited higher expression over healthy controls, which however, gradually declined when cells were cultured in vitro. Agrin expression was induced following in vitro activation of cells via their Ag receptor, or after treatment with IFN-α, a cytokine shown to be pathogenic in lupus. Furthermore, serum from SLE patients with active disease was able to induce agrin expression when added to T cells from healthy donors, an increase that was partially blocked by neutralizing anti-IFN-α Abs. Cross-linking agrin with mAbs resulted in rapid reorganization of the actin cytoskeleton, activation of the ERK MAPK cascade, and augmentation of anti-CD3-induced proliferation and IL-10 production, indicating that agrin is a functional receptor in T cells. These results demonstrate that agrin expression in human T cells is regulated by cell activation and IFN-α, and may have an important function during cell activation with potential implications for autoimmunity.

Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease involving multiple organ systems. Previous studies have suggested that interferon-lambda 1 (IFN-λ1), a type III interferon, plays an immunomodulatory role. In this study we investigated its role in SLE, including its correlation with disease activity, organ disorder and production of chemokines.

Methods

We determined levels of IFN-λ1 mRNA in peripheral blood mononuclear cells (PBMC) and serum protein levels in patients with SLE using real-time polymerase chain reaction (real-time PCR) and enzyme-linked immunoassay (ELISA). Further, we detected the concentration of IFN-inducible protein-10 (IP-10), monokine induced by IFN-γ (MIG) and interleukin-8 (IL-8) secreted by PBMC under the stimulation of IFN-λ1 using ELISA.

Results

IFN-λ1 mRNA and serum protein levels were higher in patients with SLE compared with healthy controls. Patients with active disease showed higher IFN-λ1 mRNA and serum protein levels compared with those with inactive disease as well. Serum IFN-λ1 levels were positively correlated with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), anti-dsDNA antibody, C-reactive protein (CRP) and negatively correlated with complement 3. Serum IFN-λ1 levels were higher in SLE patients with renal involvement and arthritis compared with patients without the above-mentioned manifestations. IFN-λ1 with different concentrations displayed different effects on the secretion of the chemokines IP-10, MIG and IL-8.

Conclusions

These findings indicate that IFN-λ1 is probably involved in the renal disorder and arthritis progression of SLE and associated with disease activity. Moreover, it probably plays an important role in the pathogenesis of SLE by stimulating secretion of the chemokines IP-10, MIG and IL-8. Thus, IFN-λ1 may provide a novel research target for the pathogenesis and therapy of SLE.

Excess type-I interferons (IFN-I) have been linked to the pathogenesis of systemic lupus erythematosus (SLE). Therapeutic use of IFN-I can trigger the onset of SLE and most lupus patients display upregulation of a group of interferon stimulated genes (ISGs). While this “interferon signature” has been linked with disease activity, kidney involvement, and autoantibody production, the source of IFN-I production in SLE remains unclear. Tetramethylpentadecane (TMPD)-induced lupus is at present the only model of SLE associated with excess IFN-I production and ISG expression. Here we demonstrate that TMPD treatment induces an accumulation of immature Ly6Chi monocytes, which are a major source of IFN-I in this lupus model. Importantly, they were distinct from interferon-producing dendritic cells. The expression of IFN-I and ISGs was rapidly abolished by monocyte depletion whereas systemic ablation of dendritic cells (DCs) had little effect. In addition, there was a striking correlation between the numbers of Ly6Chi monocytes and the production of lupus autoantibodies. Therefore, immature monocytes rather than DCs appear to be the primary source of IFN-I in this model of IFN-I dependent lupus.

Systemic lupus erythematosus (SLE) is a prototype systemic autoimmune disease characterized by flares of high morbidity. Using oligonucleotide microarrays, we now show that active SLE can be distinguished by a remarkably homogeneous gene expression pattern with overexpression of granulopoiesis-related and interferon (IFN)-induced genes. Using the most stringent statistical analysis (Bonferroni correction), 15 genes were found highly up-regulated in SLE patients, 14 of which are targets of IFN and one, defensin DEFA-3, a major product of immature granulocytes. A more liberal correction (Benjamini and Hochberg correction) yielded 18 additional genes, 12 of which are IFN-regulated and 4 granulocyte-specific. Indeed immature neutrophils were identified in a large fraction of SLE patients white blood cells. High dose glucocorticoids, a standard treatment of disease flares, shuts down the interferon signature, further supporting the role of this cytokine in SLE. The expression of 10 genes correlated with disease activity according to the SLEDAI. The most striking correlation (P < 0.001, r = 0.55) was found with the formyl peptide receptor-like 1 protein that mediates chemotactic activities of defensins. Therefore, while the IFN signature confirms the central role of this cytokine in SLE, microarray analysis of blood cells reveals that immature granulocytes may be involved in SLE pathogenesis.

Objective

In contrast to rheumatoid arthritis (RA), Jaccoud arthritis (JA) joint inflammation in systemic lupus erythematosus (SLE) is nonerosive. Although the mechanism responsible is unknown, the anti-osteoclastogenic cytokine interferon-alpha (IFN-α), whose transcriptome is present in SLE monocytes, may be responsible. To test this, we examined effects of IFN-α versus lupus disease on osteoclasts and erosion in the NZBxNZW F1 SLE mouse model with K/BxN serum-induced arthritis (SIA).

Methods

Elevated systemic IFN-α levels were obtained by administration of an adenoviral vector expressing IFN-α (Ad-IFN-α). SLE disease was marked by anti-dsDNA antibody titer and proteinuria, and Ifi202 and Mx1 expression represented the IFN-α transcriptome. Micro-CT was used to evaluate bone erosions. Flow cytometry for CD11b and CD11c was used to evaluate the frequency of circulating osteoclast precursors (OCP) and myeloid dendritic cells (mDC) in blood.

Results

Administration of Ad-IFN-α to NZBxNZW F1 mice induced osteopetrosis. Pre-autoimmune NZBxNZW F1 mice are fully susceptible to focal erosions in the setting of SIA. However, NZBxNZW F1 mice with high anti-dsDNA antibody titers and the IFN-α transcriptome were protected against bone erosions. Ad-IFN-α pre-treatment of NZW mice before K/BxN serum administration also resulted in protection against bone erosion (r2=0.4720, p<0.01), which was associated with a decrease in circulating CD11b+CD11c− OCP, and a concomitant increase in CD11b+CD11c+ cells (r2=0.6330, p<0.05) that are phenotypic of mDC.

Conclusion

These findings suggest that IFN-α in SLE shifts monocyte development toward mDC at the expense of osteoclastogenesis thereby resulting in decreased bone erosion.

Objective

The type I interferon (IFN) pathway is activated in many patients with systemic lupus erythematosus (SLE), and high serum levels of IFN are associated with anti-SSA/Ro autoantibodies. To investigate the clinical features associated with type I IFN production in vivo, we compared serum IFN activity in individuals with anti-SSA/Ro antibodies who were asymptomatic with that in individuals with clinical manifestations of SLE or Sjögren's syndrome (SS).

Methods

Antibody-positive sera from 84 mothers of children with manifestations of neonatal lupus were studied for type I IFN activity, using a functional reporter cell assay. Maternal health status was characterized as asymptomatic, SS, SLE, pauci-SLE, or pauci-SS, based on a screening questionnaire, telephone interview, and review of medical records. The prefix “pauci-” indicates symptoms insufficient for a formal classification of the disease.

Results

Only 4% of asymptomatic mothers had high serum type I IFN activity, compared with 73% with pauci-SLE (P = 5.7 × 10−5), 35% with SLE (P = 0.011), and 32% of patients with SS (P = 0.032). One of the 4 patients with pauci-SS had high levels of IFN. The majority of patients for whom longitudinal data were available had stable type I IFN activity over time, and changes in IFN activity were not clearly accompanied by changes in the clinical diagnosis.

Conclusion

Patients with SLE, patients with pauci-SLE, and patients with SS are more likely to have high serum IFN activity than asymptomatic individuals with SSA/Ro autoantibodies, suggesting that these autoantibodies are insufficient for activation of the type I IFN pathway, and that disease-specific factors are important for type I IFN generation in vivo.

Objective

Genetic variants of interferon regulatory factor 5 (IRF5) are associated with susceptibility to systemic lupus erythematosus (SLE). IRF5 regulates the expression of proinflammatory cytokines and type I interferons (IFN) believed to be involved in SLE pathogenesis. The aim of this study was to determine the activation status of IRF5 by assessing its nuclear localization in immune cells of SLE patients and healthy donors, and to identify SLE triggers of IRF5 activation.

Methods

IRF5 nuclear localization in subpopulations of peripheral blood mononuclear cells (PBMC) from 14 genotyped SLE patients and 11 healthy controls was assessed using imaging flow cytometry. IRF5 activation and function were examined after ex vivo stimulation of healthy donor monocytes with SLE serum or components of SLE serum. Cellular localization was determined by ImageStream and cytokine expression by Q-PCR and ELISA.

Results

IRF5 was activated in a cell type-specific manner; monocytes of SLE patients had constitutively elevated levels of nuclear IRF5 compared to NK and T cells. SLE serum was identified as a trigger for IRF5 nuclear accumulation; however, neither IFNα nor SLE immune complexes could induce nuclear localization. Instead, autoantigens comprised of apoptotic/necrotic material triggered IRF5 nuclear accumulation in monocytes. Production of cytokines IFNα, TNFα and IL6 in monocytes stimulated with SLE serum or autoantigens was distinct yet correlated with the kinetics of IRF5 nuclear localization.

Conclusion

This study provides the first formal proof that IRF5 activation is altered in monocytes of SLE patients that is in part contributed by the SLE blood environment.

Interleukin-10 (IL-10) is produced at a high level by B lymphocytes and monocytes of patients with systemic lupus erythematosus (SLE). In the present work, we analyzed whether this increased production of IL-10 contributed to the abnormal production of immunoglobulins (Ig) and of autoantibodies in SLE. The role of IL-10 was compared with that of IL- 6, another cytokine suspected to play a role in these abnormalities. The spontaneous in vitro production of IgM, IgG, and IgA by peripheral blood mononuclear cells from SLE patients was weakly increased by recombinant IL (rIL)-6, but strongly by rIL-10. This production was not significantly affected by an anti-IL-6 mAb but was decreased by an anti- IL-10 mAb. We then tested the in vivo effect of these antibodies in severe combined immunodeficiency mice injected with PBMC from SLE patients. The anti-IL-6 mAb did not significantly affect the serum concentration of total human IgG and of anti-double-stranded DNA IgG in the mice. In contrast, the anti-IL-10 mAb strongly inhibited the production of autoantibodies, and, to a lesser extent, that of total human IgG. These results indicate that the Ig production by SLE B lymphocytes is largely IL-10 dependent, and that the increased production of IL-10 by SLE B lymphocytes and monocytes may represent a critical mechanism in the emergence of the autoimmune manifestations of the disease.

The in vitro antibody response of peripheral blood lymphocytes (PBL) from 19 patients with untreated systemic lupus erythematosus (SLE) was compared with that of 20 control patients and 44 normal subjects. Trinitrophenyl polyacrylamide beads (TNP-PAA) were used to induce IgM anti-TNP plaque-forming cells. SLE patients displayed a markedly depressed, and in most instances virtually absent, response. This was not due to an unusual kinetics of the response; nor could it be induced by preincubation of SLE patients' PBL. In co-cultures of SLE patients and normal PBL, the former, with few exceptions, did not exert a suppressive effect. In four patients the anti-TNP response of either unfractionated or T-depleted SLE PBL could be restored by T cells from a normal individual. Conversely in three of these patients, SLE T cells could not support the response of normal B cells, suggesting a T helper cell defect in SLE PBL. Concanavalin A (Con A)-induced suppressor cells of the antibody response could be assayed by two approaches: (a) in responder SLE patients, by the direct addition of Con A to TNP-PAA-stimulated cultures; (b) in seven patients by transfer of Con A-activated cells to the responding culture of a normal allogeneic donor. In both cases SLE PBL were able to exert a suppressive effect to the same extent as normal PBL.

To determine whether imbalance among subsets of human T cells exists in patients with systemic lupus erythematosus (SLE), we analyzed peripheral blood lymphocytes in SLE patients during active and inactive stages of disease. For this analysis, we used monoclonal antibodies to the surface antigens of inducer (T4) and suppressor (T5/T8) T cell subsets, as well as a common T cell antigen (T3). In contrast to normal and inactive SLE patients, the percentage of T3+ cells was reduced in all active SLE patients. More importantly, there was a selective decrease in T5+/T8+ suppressor T cells in 12 of 14 active patients, including 1 of 2 patients with drug-induced SLE. Serial analysis of three SLE patients showed a significant correlation between the presence of T5+/T8+ subset and clinical disease activity in all patients. We conclude that aberrations in suppressor T cell subsets are an important correlate of disease in patients with SLE.

Among their immunological alterations patients with systemic lupus erythematosus (SLE) have been shown to have diminished natural killer (NK) cell activity. This abnormality is at least in part related to humoral factors, as sera from patients with SLE can inhibit the NK activity of peripheral blood mononuclear cells from normal individuals. The present study extends these findings to demonstrate that the inhibitory ability of sera from patients with SLE varies with disease activity. Furthermore, sera from patients with active SLE containing interferon (IFN), a potent stimulator of NK activity, were equally or more inhibitory than sera which did not contain IFN. Thus the factors in SLE sera which can inhibit NK function vary with disease activity and cannot be overcome by IFN present in these sera.

Objective

To investigate peripheral blood (PB) cell transcript profiles of systemic sclerosis (SSc) and its subtypes in direct comparison with systemic lupus erythematosus (SLE).

Methods

We investigated PB cell samples from 74 SSc patients, 21 healthy controls, and 17 SLE patients using Illumina Human Ref-8 BeadChips and quantitative polymerase chain reaction confirmation. None of the study participants were receiving immunosuppressive agents other than low-dose steroids and hydroxychloroquine. In addition to conventional statistical and modular analysis, a composite score for the interferon (IFN)–inducible genes was calculated. Within the group of patients with SSc, the correlation of the IFN score with the serologic and clinical subtypes was investigated, as were single-nucleotide polymorphisms in a selected number of IFN pathway genes.

Results

Many of the most prominently overexpressed genes in SSc and SLE were IFN-inducible genes. Forty-three of 47 overexpressed IFN-inducible genes in SSc (91%) were similarly altered in SLE. The IFN score was highest in the SLE patients, followed by the SSc patients, and then the controls. The difference in IFN score among all 3 groups was statistically significant (P < 0.001 for all 3 comparisons). SSc and SLE PB cell samples showed striking parallels to our previously reported SSc skin transcripts in regard to the IFN-inducible gene expression pattern. In SSc, the presence of antitopoisomerase and anti–U1 RNP antibodies and lymphopenia correlated with the higher IFN scores (P = 0.005, P = 0.001, and P = 0.004, respectively); a missense mutation in IFNAR2 was significantly associated with the IFN score.

Conclusion

SLE and SSc fit within the same spectrum of IFN-mediated diseases. A subset of SSc patients shows a “lupus-like” high IFN-inducible gene expression pattern that correlates with the presence of antitopoisomerase and anti–U1 RNP antibodies.

Background

It is well known that interferon (IFN)-α is important to the pathogenesis of systemic lupus erythematosus (SLE). However, several reports have indicated that the number of IFN-α producing cells are decreased or that their function is defective in patients with SLE. We studied the function of plasmacytoid dendritic cells (pDCs) under persistent stimulation of Toll-like receptor (TLR)9 via a TLR9 ligand (CpG ODN2216) or SLE serum.

Methods

The concentrations of IFN-α were determined in serum and culture supernatant of peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy controls after stimulation with CpG ODN2216 or SLE serum. The numbers of circulating pDCs were analyzed by fluoresence-activated cell sorting analysis. pDCs were treated with CpG ODN2216 and SLE serum repeatedly, and levels of produced IFN-α were measured. The expression of IFN-α signature genes and inhibitory molecules of TLR signaling were examined in PBMCs from SLE patients and healthy control individuals.

Results

Although there was no significant difference in serum concentration of IFN-α and number of circulating pDCs between SLE patients and healthy control individuals, the IFN-α producing capacity of PBMCs was significantly reduced in SLE patients. Interestingly, the degree which TLR9 ligand-induced IFN-α production in SLE PBMCs was inversely correlated with the SLE serum-induced production of IFN-α in healthy PMBCs. Because repeated stimulation pDCs with TLR9 ligands showed decreased level of IFN-α production, continuous TLR9 stimulation may lead to decreased production of IFN-α in SLE PBMCs. In addition, PBMCs isolated from SLE patients exhibited higher expression of IFN-α signature genes and inhibitory molecules of TLR signaling, indicating that these cells had already undergone IFN-α stimulation and had become desensitized to TLR signaling.

Conclusion

We suggest that the persistent presence of endogenous IFN-α inducing factors induces TLR tolerance in pDCs of SLE patients, leading to impaired production of IFN-α.

Monocytes exposed to serum from SLE patients promote B cell differentiation to IgG and IgA plasmablasts dependent on BAFF and IL-10 or APRIL, respectively.

The development of autoantibodies is a hallmark of systemic lupus erythematosus (SLE). SLE serum can induce monocyte differentiation into dendritic cells (DCs) in a type I IFN–dependent manner. Such SLE-DCs activate T cells, but whether they promote B cell responses is not known. In this study, we demonstrate that SLE-DCs can efficiently stimulate naive and memory B cells to differentiate into IgG- and IgA-plasmablasts (PBs) resembling those found in the blood of SLE patients. SLE-DC–mediated IgG-PB differentiation is dependent on B cell–activating factor (BAFF) and IL-10, whereas IgA-PB differentiation is dependent on a proliferation-inducing ligand (APRIL). Importantly, SLE-DCs express CD138 and trans-present CD138-bound APRIL to B cells, leading to the induction of IgA switching and PB differentiation in an IFN-α–independent manner. We further found that this mechanism of providing B cell help is relevant in vivo, as CD138-bound APRIL is expressed on blood monocytes from active SLE patients. Collectively, our study suggests that a direct myeloid DC–B cell interplay might contribute to the pathogenesis of SLE.

The cell surface phenotype of peripheral blood lymphocytes (PBL) of systemic lupus erythematosus (SLE) patients was characterized with the anti-2H4 monoclonal antibody that defines the human suppressor inducer subset. The T4+2H4+ population of cells has been shown to be critical for the activation of T8+ suppressor cells. Patients with SLE has a markedly decreased percentage of T4+2H4+ cells (13 +/- 2%) in their PBL compared with normal controls (21 +/- 1%) (P less than 0.001). This reduction was greatest in patients with active SLE, especially those with renal disease. Serial analysis of patients with SLE and renal disease showed a correlation between percent positive circulating T4+2H4+ cells and disease activity. Moreover, there was a significant correlation between a low percentage of T4+2H4+ cells and decreased suppressor-inducer function in autologous mixed lymphocyte reaction-activated T4+ cells from SLE patients. Thus, a deficiency exists in SLE patients with active renal disease in the T4+2H4+ suppressor-inducer T cell subset.

Introduction

Using oligonucleotide microarray, many IFN-inducible genes have been found to be highly expressed in peripheral blood mononuclear cells (PBMCs) from most patients with systemic lupus erythematosus (SLE). Among these IFN-inducible genes, IFN-induced protein with tetratricopeptide repeats 4 (IFIT4) is a novel gene whose function is unknown.

Methods

In this study we examined the role played by IFIT4 in monocyte differentiation and the correlation between IFIT4 expression and the clinical manifestation of SLE. To this end, we used plasmid transfection, flow cytometry, mixed leucocyte responses, ELISA, quantitative RT-PCR and Western blotting.

Results

We found that both IFIT4 mRNA and protein expression levels were significantly higher in PBMCs and monocytes from SLE patients than in those from healthy control individuals. IFIT4 expression was positively correlated with antinuclear antibodies, anti-double-stranded DNA, and anti-Sm auto-immune antibodies in SLE. Patients with SLE exhibiting higher expression of IFIT4 had a higher prevalence of leucopenia, thrombocytopenia and C3/C4 decrease. IFIT4 protein was localized exclusively to the cytoplasm, and it was significantly upregulated by IFN-α in normal PBMCs. To determine the role played by IFIT4 in monocyte differentiation, the monocytic cell line THP-1 was transfected with pEGFP-IFIT4 expression plasmid and stimulated with granulocyte-macrophage colony-stimulating factor/IL-4 to generate IFIT4-primed dendritic cell-like cells (DCLCs). IFIT4-primed DCLCs acquired morphological characteristics of dendritic cells more quickly, with greater resemblance to dendritic cells, as compared with DCLCs primed with pEGFP-C1 control plasmid trasfection. Furthermore, they exhibited higher expressions of CD40, CD86, CD80, HLA-DR and CD83, along with lower expression of CD14; increased IL-12 secretion; and an increased ability to stimulate T-cell proliferation. In addition, IFIT4-primed DCLCs enhanced IFN-γ secretion (about 2.4-fold) by T cells compared with controls.

Conclusion

Our findings suggest that IFIT4 might play roles in promoting monocyte differentiation into DCLCs and in directing DCLCs to modulate T-helper-1 cell differentiation; these actions might contribute to the autoimmunity and pathogenesis of SLE.

The interferon (IFN)-induced enzyme 2-5A synthetase was elevated in mononuclear cells from both serum IFN-positive and -negative systemic lupus erythematosus (SLE) patients. This suggests that a much higher percentage of patients than previously thought produce endogenous IFN. These results may partly explain findings that mononuclear cells from SLE patients are deficient in IFN production in vitro in response to certain IFN inducers. Although normal lymphocytes can produce an acid- labile alpha IFN after stimulation with C. parvum in vitro, the reason for endogenous production of this unusual alpha IFN by SLE patients remains unknown.